A growing body of evidence suggests a pathogenic role for pro-inflammatory T helper 17 cells (Th17) in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, and psoriasis - diseases for which no curative treatment is currently available. Differentiated Th17 cells are characterized by the production of pro-inflammatory cytokines including IL-17A (IL-17), IL-17F, IL-21, and IL-22. Two members of the Retinoid-related Orphan Receptor (ROR) subfamily of Nuclear Hormone Receptors have been identified as key regulators of Th17 differentiation: RORgammat (a thymus-specific isoform of RORgamma with identical Ligand Binding Domain) and RORalpha. Targeting Th17 cell differentiation by inhibiting RORgammat and/or RORalpha has thus become an attractive strategy for potential treatment of inflammatory autoimmune diseases. Fine tuning inhibition of these two receptors may be desirable to minimize safety risks, as IL-17 is important for controlling host defenses against bacterial infections and complete elimination of IL-17 could be associated with increased risk of infection. RORs have been orphan receptors until very recently. Once the crystal structure of RORalpha revealed the presence of cholesterol in the ligand binding pocket, biochemical and structural studies established that hydroxycholesterols function as natural ligands for RORgamma. In addition, two natural products (digoxin and ursolic acid) and one synthetic molecule (SR1001), which were found to function as RORgamma antagonists, are effective in suppressing Th17 differentiation and delaying the onset of disease in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. This proves the efficacy of RORgamma inhibitors as a potential therapeutic strategy for autoimmune diseases, and encourages further research to discover novel compounds with enhanced potency and selectivity that could offer better clinical alternatives. We have identified novel chemical entities that function as RORgamma antagonists from Mixture-based Synthetic Combinatorial Libraries. Our goals are i) to evaluate the effect of our lead compounds in Th17 cell differentiation and autoimmune disease in mice; ii) to further deconvolute active mixtures following a cell-based screening platform to identify novel negative modulators of RORgamma activity; iii) to establish how the ROR- selectivity profile of these novel RORgamma inhibitors affects Th17 differentiation and autoimmune disease; and iv) to optimize leads following a structure-based approach to improve their potency and RORgamma selectivity. TPIMS mixture-based chemical libraries are unique and offer an excellent source for innovative RORgamma ligands. Based on our preliminary studies and our experience with this type of chemical libraries, we are confident that this proposal will produce novel potent RORgamma antagonists that will prove effective in preventing Th17 cell differentiation and delaying the onset and/or ameliorating autoimmune disease in mice. These compounds will offer an exceptional opportunity for the development of innovative RORgamma-based therapeutics against inflammatory autoimmune diseases.